1. Field of the Invention
This invention relates to an improved process for the production of cephalosporin C. More particularly, the invention relates to the addition of certain organic and inorganic phosphorous compounds to the culture medium during fermentation of a cephalosporin C-producing microorganism which also produces undesired desacetylcephalosporin C. Addition of the phosphorous compounds greatly inhibits formation of the desacetylcephalosporin C impurity, thus facilitating recovery of the cephalosporin C from the fermentation broth and its subsequent conversion to 7-aminocephalosporin acid (7-ACA).
2. Description of the Prior Art
Cephalosporin C [3-acetoxymethyl-7.beta.-(D-5-amino-5-carboxypentanamido)ceph-3-em-4-carbo xylic acid] is a compound which, while having some antibiotic activity per se, is of primary importance as a starting material for preparation of semi-synthetic cephalosporin antibiotics. Thus, cephalosporin C may be converted by known methods to 3-acetoxymethyl-7.beta.-aminoceph-3-em-4-carboxylic acid (7-ACA) which is then used as a key intermediate for preparation of a wide variety of commercial cephalosporin antibiotics.
It is known that cephalosporin C may be obtained by fermentation of various microorganisms including especially fungi of the genera Emericellopsis-Cephalosporium. Illustrative of cephalosporin C-producing microorganisms are the original Brotzu strain of Cephalosporium, i.e. Cephalosporium sp. I.M.I. 49137 (ATCC 11550), and mutants thereof such as mutant strain 8650 (ATCC 14553), described in U.K. Pat. No. 1,109,362, Cephalosporium sp. I.B.I. 1131 described in U.K. Pat. No. 1,503,851 and Cephalosporium sp. strain F.12 (ATCC 20339) described in U.K. Pat. No. 1,400,433. Other examples of cephalosporin C-producing organisms reported in the literature include Cephalosporium polyaleurum Y-505 (FERM-P No. 1160) described in U.K. Pat. No. 1,488,822, Cephalosporium acremonium K-121 (ATCC 20427) and Cephalosporium acremonium N-75 (ATCC 20428) described in U.K. Pat. No. 1,488,821 and Cephalosporium polyaleurum 199 (ATCC 20359) and a mutant thereof identified as Y-505 (ATCC 20360) described in U.K. Pat. No. 1,389,714. Cephalosporin C is generally produced on an industrial scale by use of a high-producing mutant strain of Cephalosporium acremonium (also known as Acremonium chrysogenum). Examples of such mutants and methods for their preparation have been extensively described in the literature.
Despite exhaustive research over the years, fermentation of cephalosporin C on a commercial scale is still not entirely satisfactory. Most cephalosporin C-producing microorganisms, especially those high producing strains used in commercial production, result in co-production of a significant proportion of desacetylcephalosporin C, an impurity which is extremely difficult to separate from the desired cephalosporin C product because of its similar chemical and physical characteristics. Presence of the desacetylcephalosporin C, typically in amounts of about 15% of the total cephalosporin nucleus produced during fermentation, results in recovery of cephalosporin C (or more commonly, a solvent-extractable derivative thereof) contaminated with desacetylcephalosporin C (or derivative thereof). Moreover, since on an industrial scale the cephalosporin C (or derivative thereof) is usually not purified prior to subsequent conversion to 7-ACA, product quality of the 7-ACA is also adversely affected by the concomitant production of desacetylcephalosporin C in the initial fermentation broth.
The prior art dealing with cephalosporin C production is primarily concerned with finding new microorganisms of higher cephalosporin C productivity and providing fermentation additives which increase cephalosporin C production. Thus, for example, mutant strains of Cephalosporium acremonium have been developed which produce substantially higher yields of cephalosporin C. It has been suggested to add various additives to the nutrient medium during fermentation of a cephalosporin C-producing organism so as to increase the cephalosporin C yield. Thus, the use of sulfur compounds such as sodium sulfite, sodium metabisulphite, sodium thiosulfate, sodium hydrosulphite, sodium thiosulphate and sodium sulphate are disclosed in U.K. Pat. No. 820,422, use of methionine, calcium chloride, magnesium chloride, ammonium sulfate and certain carbohydrates, oils and fatty acids is disclosed in U.K. Pat. No. 938,755, use of norvaline and norleucine is disclosed in U.K. Pat. No. 975,393, use of phenylacetic acid is disclosed in U.K. Pat. No. 975,394 and use of .epsilon.-caprolactam, 2-butanone, secondary butyl alcohol and 1,3-butanediol is disclosed in U.K. Pat. No. 1,503,851. The problem of co-production of desacetylcephalosporin C during cephalosporin C fermentation has been addressed only in terms of providing microorganisms which produce higher proportions of cephalosporin C nucleus as cephalosporin C or in terms of extraction/isolation procedures (e.g. U.S. Pat. No. 4,059,573).
Desacetylcephalosporin C was first detected in culture filtrates of Cephalosporium acremonium. Abraham et al. proposed that formation of this substance was due to the enzymatic deacetylation of cephalosporin C (Biochem. J. 81: 591-596, 1961). Subsequently, esterase enzymes capable of deacetylating cephalosporin C have been isolated from a variety of sources, for example, citrus fruits, bacteria, actinomycetes, wheat germ, mammalian liver and kidney and Rhodotorula. Pisano et al. in Develop. Ind. Microbiol. 8: 417-423, 1967, report that esterase activity is widespread in the genus Cephalosporium. The majority of these acetylesterase enzymes appear to have broad substrate tolerances, i.e. .beta.-naphthyl acetate and triacetin are active substrates, and their activity toward cephalosporin C does not appear unique.
Nuesch et al. in Second International Symp. on Genetics of Industrial Microorganisms, Proc., 1975, ed. MacDonald, K. D., New York, Academic Press, pg. 451-472 and Fujisawa et al. in Agr. Biol. Chem. 39(6): 1303-1309 (1975) independently partially purified cephalosporin C esterase activity from extracellular broth supernatant of Cephalosporium acremonium and concluded that the presence of the enzyme activity was partially responsible for the occurrence of desacetylcephalosporin C in C. acremonium fermentations. Similar esterase activity has been detected in the cephalosporin C producing Streptomycetes Streptomyces clavuligerus (Antimicrob. Agents Chemother. 1: 237-241, 1972). Huber in Appl. Microbiol. 16(7): 1011-1014 (1968), however, has presented evidence that the formation of desacetylcephalosporin C during the fermentation process is due to the non-enzymatic hydrolysis of cephalosporin C. It is the opinion of the present inventors that desacetylcephalosporin C formation is due to both enzymatic and non-enzymatic hydrolysis, with enzymatic acetylesterase activity playing a significant role.
Reports by Liersch et al. in Second International Symp. on Genetics of Industrial Microorganisms, Proc., 1976, ed. MacDonald, K. D., New York, Academic Press, pg. 179-195 and Felix et al. in FEMS Microbiol. Lett. 8: 55-58, 1980 have indicated that desacetylcephalosporin C is also an intracellular intermediate in the biosynthesis of cephalosporin C from desacetoxycephalosporin C.
The enzyme activity of the partially purified acetylesterase from Cephalosporium acremonium was reported to be inhibited by diisopropylfluorophosphate, a recognized inhibitor of esterases (Agr. Biol. Chem. 39(6): 1303-1309, 1975). The extreme toxicity and high cost of this phosphorous acetylesterase inhibitor, however, prevents its use in the commercial production of cephalosporin C.
Cephalosporin C, because of its amphoteric nature, is normally converted into a derivative so that it can be more easily recovered from the fermentation broth by solvent extraction procedures. Examples of such derivatives are given in U.K. patent application No. 2,021,640A. One particularly preferred process is disclosed in U.S. Pat. No. 3,573,296. The cephalosporin C derivative obtained by such preferred process may be recovered as a crystalline bis-dicyclohexylamine salt as disclosed in U.S. Pat. No. 3,830,809. The cephalosporin C or derivative thereof recovered from the fermentation broth is then cleaved by a conventional procedure, e.g. the process of U.S. Pat. No. 3,932,392, to provide 7-ACA.
As noted above, the desacetylcephalosporin C impurity typically obtained during fermentation in amounts of about 15% of the total cephalosporin nucleus (cephalosporin C and desacetylcephalosporin C) has chemical and physical characteristics quite similar to those of the desired cephalosporin C product. Thus, when the cephalosporin C is converted to a solvent-extractable derivative, the desacetylcephalosporin C is also converted to a similar derivative and the cephalosporin C derivative then isolated is contaminated with the desacetylcephalosporin C derivative. It can be seen, therefore, that reducing the proportion of cephalosporin nucleus obtained as desacetylcephalosporin C will result in a purer cephalosporin C derivative product. Moreover, since this derivative is not normally purified prior to conversion to 7-ACA, reduced amounts of desacetylcephalosporin C in the fermentation broth will also result in a better quality 7-ACA product.
The present invention is directed toward provision of certain phosphorous compounds which act as inhibitors of desacetylcephalosporin C production during fermentation of cephalosporin C. The resulting fermentation broth contains a significantly higher proportion of cephalosporin C to desacetylcephalosporin C, thus improving the quality of the recovered cephalosporin C product and, in turn, the quality of the ultimate 7-ACA intermediate prepared from such cephalosporin C product.